A kind of staphylococcus protein a, purification preparation method and application thereof

A staphylococcus protein, staphylococcus technology, applied in the preparation method of peptide, biochemical equipment and method, immunoglobulin and other directions, can solve the problems of insufficient purity and insufficient purity of IgG, achieve high purification efficiency, simple preparation process, High alkali resistance effect

Active Publication Date: 2022-08-09
赣江中药创新中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein A of this structure binds a large amount of IgG, while its non-Fc binding domain can bind some miscellaneous proteins, resulting in insufficient purity of the eluted IgG. Generally, the purity of industrially synthesized antibodies can exceed 90% after one-step protein A affinity chromatography. %
But for monoclonal antibodies, this purity is often not enough

Method used

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  • A kind of staphylococcus protein a, purification preparation method and application thereof
  • A kind of staphylococcus protein a, purification preparation method and application thereof
  • A kind of staphylococcus protein a, purification preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0030] A staphylococcal protein A, the amino acid sequence of the B functional domain of the staphylococcal protein A is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0031] A kind of purification preparation method of staphylococcus protein A, described method comprises

[0032] Partial site mutation of the B functional domain of Protein A protein derived from Staphylococcus to form Protein A-1 and Protein A-2;

[0033] A recombinant plasmid was constructed, the prokaryotic system induced expression, and the expressed recombinant Protein A protein was separated and purified by Ni affinity chromatography and ion exchange chromatography, and the purity was greater than 95%.

[0034] In this example, the mutation sites of Protein A-1 include: A176V, N178H, N181D, Q184A, E190K, N198T, N203G, G204A, N218A, A221S, D228E, A229S, a total of 12 mutations, the amino acid sequence is as shown in SEQ IDNO: 1.

[0035] In this example, the mutation sites of Protein A-2 include: A176V, Q184H...

Embodiment 2

[0041] 1.1 Protein A protein gene synthesis modification design:

[0042] The mutation sites include: A176V, N178H, N181D, Q184A, E190K, N198T, N203G, G204A, N218A, A221S, D228E, A229S protein A-1 protein with a total of 12 mutations, and A176V, Q184H, E190K, N198S, N203G , G204A, D211H, N218A, A221S, D228E, A229S protein A-2 protein with a total of 11 mutations, the purpose is to enhance the stability and alkali resistance of the target protein.

[0043] Protein recombinant plasmid construction:

[0044] A protein A protein purification preparation method and application of the present invention, according to the gene synthesis transformation design in 1.1, entrust GenScript Company to synthesize the corresponding gene, and connect it to the pET30a (GenScript) vector to construct a recombinant plasmid. In order to better illustrate the superior performance of Protein A of the present invention, wild-type Protein A (WT) was also commissioned to be synthesized by a company as ...

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Abstract

The invention discloses a staphylococcus protein A, a purification preparation method and application thereof. The invention mainly relates to the construction of a recombinant plasmid by linking a genetically engineered protein ProteinA gene into an expression vector, and then introducing it into a prokaryotic host cell for expression. And and ion exchange chromatography purification, can obtain the target protein with a purity of more than 95%. The purification preparation method of the invention has the characteristics of simple purification process and high yield. At the same time, the prepared ProteinA has good immunoglobulin IgG binding ability, alkali resistance and thermal stability. In addition, the affinity medium prepared by the solid-phase carrier coupled with ProteinA has the advantages of good IgG adsorption effect, high sample loading capacity and alkali resistance. The ProteinA protein prepared by the invention and the medium prepared by coupling can play an important role in the field of monoclonal antibody purification and immunodiagnosis research.

Description

technical field [0001] The invention relates to the technical field of cloning and transforming Protein A in genetic engineering, in particular to a staphylococcus protein A, a purification preparation method and applications thereof. Background technique [0002] In the mid-20th century, Jensen discovered a protein that was first known as Protein A in 1964, with broad binding to antibodies from human and rabbit serum. Protein A is a Staphylococcus aureus cell wall protein with a molecular weight of 42KDa, which can specifically bind to the Fc region of a variety of immunoglobulins but weakly bind to the Fab region or light chain. Natural Protein A has 5 IgG binding domains E, D, A, B, C and non-Fc binding domains of unknown function. 5 different domains can bind to the Fc fragment of IgG and have strong specific affinity. Each Protein A molecule can bind at least two IgG molecules. At the same time, IgA, IgM or IgE may also bind to Protein A. While the protein A of this ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/31C12N15/70C07K1/18C07K16/00C07K1/22G01N33/53B01J20/281C12R1/19
CPCC07K14/31C12N15/70C07K16/00C07K1/22G01N33/53B01J20/281Y02A50/30
Inventor 刘龙英叶贤龙郭志谋于伟高岩华熊京京徐思梦胡文峰
Owner 赣江中药创新中心
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